Production of fatty acid estolides

ABSTRACT

It has been found that esterification of a hydroxy-fatty acid by a lipase can be coupled with oleate hydratase (OHase) generation of that hydroxy-FA from an unsaturated FA with a cis C9-C10 double bond, e.g. oleic acid, in a single aqueous buffered reaction medium at low temperature, e.g. 30° C. A simple one-pot enzymatic method to produce fatty acid estolides from one or more triglycerides, e.g. starting from a natural plant oil, is thereby enabled in which the same lipase catalyses both the initial hydrolysis of triglyceride and the final esterification step.

RELATED APPLICATION DATA

This application is a divisional of U.S. patent application Ser. No.15/561,644, filed Sep. 26, 2017, which is the national phase ofInternational Application No. PCT/EP2016/056656, filed Mar. 24, 2016,which claims priority to United Kingdom Application No. 1505178.2, filedMar. 26, 2015, and to Provisional Application No. 62/138,541, filed Mar.26, 2015.

FIELD OF THE INVENTION

The present invention relates to production of fatty acid estolides,more particularly estolides derived from oleic acid and otherunsaturated fatty acid substrates for oleate hydratase (OHase) whichhave a cis double bond between C9 and C10. Oleate hydratase convertssuch substrates to 10-hydroxy fatty acids. Thus oleic acid, one of themost abundant, natural, unsaturated fatty acids, is converted by oleatehydratase to 10-hydroxystearic acid (10-HSA); see FIG. 1. The resulting10-hydroxy fatty acids can be used to form estolides in which thecarboxylic acid functionality of one fatty acid chain forms a secondaryester linkage to the alkyl backbone of another. For example, 10-HSA canform a monoestolide with oleic acid and esters with itself.

It has now been found that formation of such estolides employing 10-HSAand other 10-hydroxy fatty acids can be catalysed by a lipase underaqueous conditions suitable for oleate hydratase activity, i.e. forexample in aqueous buffered solution at about pH 6-6.5 and at lowtemperature, e.g. 30° C. It has further been shown that consequently itis feasible to couple fatty acid estolide formation with oleatehydratase activity on an unsaturated fatty acid substrate, e.g. oleicacid, or both oleate hydratase activity on such a substrate and lipasehydrolysis of a triglyceride, e.g. triolein, in a single reactionmixture whereby the monomers for estolide formation are provided in situin the reaction mixture. This opens up for the first time thepossibility of a simple one-pot enzymatic process to produce esters of10-hydroxy-fatty acids from natural oils or a purified triglyceridepreparation using two enzymes to catalyse three consecutive reactions—asingle lipase to catalyse both the hydrolysis of a triglyceridesubstrate (1st reaction) and ester synthesis (3rd reaction) coupled witha OHase. Fatty acid estolides can advantageously thereby be providedfrom bio-based feedstock, e.g. natural plant oils, for many industrialapplications including use as biodegradable lubricants and coatings andfor use in the food and cosmetic industries (Cermak & Isabell (2009)Ind. Crops Products 29, 205-212).

BACKGROUND TO THE INVENTION

Conventional chemical synthesis of fatty acid estolides from unsaturatedfatty acids uses high temperatures, generally above 200° C., and aninorganic catalyst such as tin, titanium or sulphuric acid, usually indry organic media. Such processes can lead to degradation of the esterand undesired side reactions. Additionally, the resulting energy costsare high. With a view to overcoming such problems, attention ofresearchers has moved to use of lipases as biocatalysts forhydroxy-fatty acid esterification (Martin-Arjol et al. (2013) ProcessBiochemistry 48, 224-230).

Lipases have long been known for hydrolysing the triglyceride componentsof oils to release the fatty acid component(s), e.g. release oleic acidfrom triolein in which glycerol is esterified with three oleic acidchains. Such enzymatic hydrolysis is usually performed in an aqueoussolution that is contacted with the oil forming a liquid-liquiddispersion at ambient conditions (typically 35° C. and atmosphericpressure). The lipase catalyses the hydrolysis of the triglyceridecomponent(s) at the interface between the two liquids. The reaction isreversible so that the final composition of the products and thehydrolysis rate depends on the fatty acid concentration in the oil phaseand on the glycerol concentration in the water phase. Use of immobilizedlipase is also known for this purpose (Ramachandra et al. (2002)Biotechnol. Bioprocess Eng. 7, 57-66). The main constituents of plantoils are triacylglycerols and hence they are recognised as an importantrenewable source of fatty acids for various synthetic uses through suchlipase action (De Espinosa & Meier (2011) European Polymer J. 47,837-852: ‘Plant oils: The perfect renewable resource for polymerscience?!’).

Though designed by nature to effect hydrolysis of triglycerides, it hasalso long been recognised that lipases can, under appropriateconditions, promote esterification as required for estolide formation(F. D. Gunstone (1999) J. Sci. Food Agric. 79, 1535-1549). Much researchin relation to such lipase use has focused on formation of estolidesfrom naturally-occurring mono-hydroxy-fatty acids such as ricinoleicacid (12(R)-hydroxy-9(Z)-octadecanoic acid). Castor oil has a highpercentage of triglyceride containing ricinoleic acid and hence is wellknown as a source of commercially available ricinoleic acid obtained byconventional saponification.

In 1989, Yamaguchi et al. reported the direct production of fatty acidestolide from castor oil by provision of a lipase in an aqueousdispersion of castor oil at 30-65 wt %. A good yield of estolide wasobtained with use of a lipase capable of hydrolysing at the glyceridebeta site or a lipase having partial glyceride selectivity, but not witha lipase having selectivity for the glyceride alpha site. The relevantstudies are reported in published Japanese patent applicationsJPS64-16591 and JPS64-16592. However, such estolide formation relying onthe C12 hydroxy group of naturally-occurring ricinoleic acid is notsuggestive of lipase-catalysed estolide formation from any C10hydroxy-fatty acid under conditions compatible with function of anoleate hydratase to provide the hydroxy-FA in situ. Yamaguchi et al. hadno need to consider maintaining activity of an oleate hydratase and werelimited to consideration of oils that contain a high amount ofricinoleic acid. Moreover, later work of the same group focused on useof immobilised lipase for estolide formation from the same hydroxy-FA;they suggested as preferable use of immobilized Candida rugosa lipase ona ceramic carrier with removal of water to carefully control the watercontent in the reaction environment; see Japanese Patent no. 3157028 andYoshida et al. (1997) JAOCS 74, 261-267.

More recently, published European Patent Application no 2757158(Petroleo Brasileiro SA), and the equivalent published US PatentApplication US2013102041A, proposed enzymatic synthesis of fattyestolides using an immobilised microbial lipase non-specific for the1,3-positions of a triglyceride, such as the lipase of Candida rugosa orderived from a Pseudomonas species, in a solvent-free medium at 70-90°C. and with maintenance of a very low water concentration. Stearic acidand methylricinoleate (biodiesel from castor oil) were employed forestolide formation using the immobilized form of recombinant lipase B ofCandida antarctica commercially available as Novozyme 435. Similarstudies by the same group looking at estolide formation by immobilizedlipase from oleic acid and methylricinoleate are discussed in Aguieraset al. (2011) Enzyme Research, Article ID. 432746.

Others have also looked at various operating conditions to improvericinoleic estolide formation using immobilized lipase in solvent-freesystems with emphasis on control of water concentration, e.g. use ofmolecular sieves to adsorb water and vacuum reaction with air-drying(see for example Bódalo et al. (2009) Biochem. Eng. J. 44, 214-219;Horchani et al. J. Mol. Catal B: Enzym (2012) 75, 35-42). More recently,lipase synthesis of esters of hydroxyl-fatty acids in dry organicsolvents such as n-hexane has been proposed (Martin-Arjol et al. (2013)ibid). Such systems are not compatible with any consideration ofcoupling of lipase-synthesis of fatty acid estolides with oleatehydratase production of hydroxy-FA in the same reaction medium.

Hayes and Kleinman (1995) JAOCS 72, 1309-1316 reports screening of anumber of microbial lipases for ability to form estolides from thenaturally occurring cis unsaturated hydroxy-FA known as lesquerolic acid(14(R)-hydroxy-11(Z)-eicosenoic acid) and oleic acid in differentreaction systems: native lipases in aqueous-organic biphasic mediumemploying isooctane, immobilised lipase and reverse micelles. Theresults further support that ‘random lipases’, i.e. lipases lacking1,3-positional specificity with respect to triglyceride (for example thelipases of Candida rugosa, Chromobacterium viscosum, Geotrichum candidumand Pseudomonas species) are effective in catalysing estolide formation.In contrast, 1,3-positional specific lipases tested were ineffective(with the exception of Aspergillus niger lipase which provided a smallamount of estolide). This accords with such lipases not utilising, orpoorly utilising, secondary alcohols as substrate. The productdistribution was also found to be dependent on the lipase source. Use ofCandida rugosa lipase or Geotrichum lipase gave estolide formation withgreater than 80% of the estolide being the monoestolide formed from onelesquerolic acyl group and one octadecanoic acyl group. Pseudomonas sp.lipase gave a very different product mixture including a significantamount of monoestolide with two lesquerolic acyl groups and somediestolide. However, again no information is provided relevant tocoupling lipase esterification with formation of a hydroxy-FA by oleatehydratase.

Todea et al (2015) Pure Appl. Chem. 87, 51-58 reported the screening ofa number of free and immobilized lipases and an immobilized protease fortheir capability to synthesize estolides form hydroxy acids with primaryand secondary hydroxyl groups, i.e. 16-hydroxyhexadecanoic acid(16-HHDA), 12-hydroxy-9-cis-hexadecenoic acid (ricinoleic acid, RCA),and 12-hydroxy-octadecanoic acid (12-HSA), in dry organic solvents at60° C. The results showed that non-regiospecific lipases are able tocatalyze the synthesis of estolides from both primary and secondaryhydroxy-fatty acids; the reactivity of lipases for the tested substratesdecreased in the order: C16(16OH)>C18(12OH:9)>C18(12OH). Most efficientwere found to be lipases from Pseudomonas fluorescens, Pseudomonas.stutzeri, C. antarctica B, C. rugosa, Alcaligenes species andThermomyces lanuginosus. The product distribution depended on the typeof substrate and the type of lipase, but the main product in all caseswas the monoestolide, representing 60-80% of the product formed. LipasesB from C. antarctica and Pseudomonas lipases produced longer chainestolides including di-, tri- and tetramers, while small amounts ofmonolactones (i.e. cyclic esters derived from the hydroxy-fatty acid byring closure) where obtained only with lipase from P. fluorescens and P.stutzeri.

As part of the same studies, various immobilized lipases (lipases ascross-linked enzyme aggregates (CLEA) from P. stutzeri (CLEA-P. stutzen)and C. antarctica B (CLEA CaIB) and CLEA Alcalase, T. lanuginosus lipaseimmobilized on granulated silica (Lipozyme TL) and C. antarctica lipaseimmobilized on acrylic resin (Novozyme 435) were found to achieve highyields of estolides, when incubated with the same hydroxy-fatty acids at75° C. for 24 hours in toluene. The main product obtained was themonoestolide. While longer estolides were obtained with all enzymes and16-HHDA containing a primary hydroxyl group, immobilized enzymesproduced only the monoestolide of 12-HSA and ricinoleic acid with theexception of the CLEA-P. stutzeri lipase that produced a mixture ofpolyestolides up to heptamers for 12-HSA and decamers for ricinoleicacid.

Estolides have been found to be detectable in submerged cultures ofPseudomonas aeruginosa 42A2 when cultivated with oleic acid and whenoleic acid is incubated with partially purified lipase obtained from thesame Pseudomonas for 72 hours at 30° C. in a Tris buffer mediumcontaining sodium deoxycholate and CaCl₂. However, the pH of thereaction medium was 9.2 and such estolide formation has solely beenlinked with biotransformation of oleic acid to two trans unsaturatedhydroxy-fatty acid derivatives by a reaction mechanism unrelated tooleate hydratase (Pelàez et al. (2003) JAOCS 80, 859-866; Guerrero etal. (1997) Biochem. Biophys. Acta 1347, 75-81; Martinez et al. (2010) J.Biol. Chem. 285, 9339-9345; Martin-Arjol et al. (2013) Ibid.)

The biotransformation of oleic acid into 10-hydroxystearic acid wasfirst identified in a presumed Pseudomonas strain, designated as strain3266 more than half a century ago (Wallen et al. (1962) Arch. Biochem.Biophys. 99, 249-25). Due to its properties such as good fluidity at lowtemperature and good oxidative stability, oleic acid was subsequentlysuccessfully tested as a substrate for microbial hydration by a varietyof microorganisms including other Pseudomonas sp. strains and species ofNocardia (Rhodococcus), Corynebacterium, Sphingobacterium, Micrococcus,Macrococcus, Aspergillus, Candida, Mycobacterium andSchizosaccharomyces. The product stereospecificity is dependent on themicroorganism. For example, mixtures of enantiomers were obtained usingRhodococcus rhodochrous ATCC 12674 and Sphingobacterium, while opticallypure 10(R)-HSA resulted by using Pseudomonas sp. NRRL B-3266 (See Hou(2009) New Biotechnology 26, 105-108, ‘Biotechnology for fats and oils:new oxygenated fatty acids’).

In 2009, Bevers et al. first reported isolation and biochemicalcharacterisation of the oleate hydratase from Pseudomonas strain 3266,re-classified on the basis of sequence information as Elizabethkingiameningoseptica. The coding sequence for the OHase was cloned (GenBankaccession no. GQ144652) and expressed in E. coli as an N-terminal Histag protein to investigate the activity of the OHase in different pHbuffers. The optimal pH for activity was observed to be around pH 6; atpH 9 no significant activity was observed. The presence of salt (NaCl)was also shown to influence activity. With the method employed forrecombinant OHase production, an optimal concentration of 50 mM NaCl wasfound in 20 mM Tris buffer (pH 8) at 22° C. It was also observed thatthe specific activity of the enzyme did not change significantly in thepresence of 2.5% or 5% isopropyl alcohol.

However, it decreased at isopropyl concentrations higher than 10%(Bevers et al. (2009) J. Bacteriol. 191, 5010-5012).

A recombinantly produced fatty acid hydratase of Streptococcus pyrogenesreported in published International Application WO2008/119735(Georg-August-Universität) was subsequently identified as a homologue ofthe E. meningoseptica OHase (Bevers et al. (2009) ibid). In 0.1M sodiumphosphate buffer at pH 7.1 containing 0.1 M NaCl, it was shown at 37° C.to act preferably at the cis-9 double bond of oleic acid but also to beable to convert other unsaturated fatty acids with a cis C9-C10 doublebond (palmitoleic acid, linoleic acid and α-linolenic acid) to thecorresponding 10-hydroxy-fatty acid.

A number of other microbial oleate hydratases have since been studied asrecombinant functional enzymes. For example, Kim et al. (2012) Appl.Microbiol. Biotechnol. 95, 929-937 reports studies on the production of10-HSA from oleic acid and olive oil hydrolysate using an oleatehydratase of Lysinibacillus fusiformis expressed in E. coli. The optimalreaction conditions for producing 10-HSA were found to be aqueous bufferat pH 6.5, 35° C., 4% (v/v) ethanol. The hydration activity was shown tobe highest for oleic acid but activity was also shown against a numberof other unsaturated fatty acids of length C14 to C18 with a cis C9-C10double bond: myristoleic acid (C14), palmitoleic acid (C16), linoleicacid (C18), α-linolenic acid (C18) and γ-linolenic acid (structuresshown in FIG. 2). The same OHase was subsequently shown to act also atthe C9-C10 cis double bond of ricinoleic acid under similar conditions(pH 6.5, 30° C., 4% (v/v) methanol) to give the dihydroxy fatty acid10,12-dihydroxystearic acid (Seo et al. Appl. Microbiol. Biotechnol.(2013) 97, 8987-8995).

Conversion of oleic acid to 10-HSA using whole cells of recombinant E.coli expressing a microbial OHase has also been reported. Thus, Joo etal. (2012) J. Biotechnol. 158, 17-23 reports conversion of oleic acid to10-HSA by recombinant E. coli expressing the OHase of Stenotrophomonasmalltophilia at pH 6.5 and 35° C. with 0.5% (w/v) Tween 40.

The same group has also studied the oleate hydratase of Macrococcuscaseolyticus obtained by recombinant expression in E. coli (Joo et al.(2012) Biochemie 94, 907-915). Maximum activity against oleic acid wasobserved at pH 6.5 and 25° C. with 2% (v/v) ethanol and 0.2 mM FAD. Thehydratase was shown to be FAD-dependent; in the absence of FAD nocatalytic activity was observed. Besides the activity to oleic acid, thesame hydratase was also found to convert myristoleic acid to10-hydroxytetradecanoic acid and palmitoleic acid to10-hydroxyhexadecanoic acid respectively with no by-products. Tworeactions were observed when linoleic acid, α-linolenic acid andγ-linolenic acid were used as substrates due to additional hydration atthe available cis-12 double bond to give a dihydroxy fatty acid.

Thus linoleic acid gave both 10-hydroxy-12(Z)-octadecanoic acid and10,13-dihydroxy-octadecanoic acid. α-Linolenic acid gave both10-hydroxy-12(Z),15(Z)-octadecadienoic acid and10,13-dihydroxy-15(Z)-octadecenoic acid and γ-linolenic acid gave both10-hydroxy-6(Z),12(Z)-octadecadienoic acid and10,13-dihydroxy-6(Z)-octadecanoic acid.

More recently, the inventors for the present application have reporteddemonstration of total conversion of oleic acid to 10-HSA usingrecombinant oleate hydratase of Elizabethkingia meningoseptica producedby E. coli as either a free enzyme or immobilized (poster presentationat Biotrans 2013). Purified OHase is recognised to be a very unstableenzyme; with simple storage in buffer at 4° C., recombinant oleatehydratase of E. meningoseptica has been found by the inventors to loseabout 60% of its activity in 7 days. It was also shown however that boththe thermal stability and stability with re-use (operational stability)can be significantly enhanced by immobilization. Of especial note wasthe finding that among various immobilization methods investigated,covalent linkage on to magnetic chitosan composite particles results inparticularly effective stabilization during multiple reuses with enzymerecovery by magnetic separation; the covalently bound enzyme preserved75% of its initial activity after five reuses at 30° C. for 2 hours percycle. Furthermore, the same immobilized OHase showed improved thermalstability; it retained more than 65% of its original activity afterincubation at 50° C. (reported by oral presentation at ProtStab 2014 andNetherlands Chemistry and Catalysis Conference NCCC 2015).

Heo et al. investigated the ability of Flavobacterium sp strain DS5(NRRL B-14859) to directly convert olive oil and soybean oil tooxygenated fatty acids such as 10-ketostearic acid and 10-HSA. Lipaseaddition to the culture medium was required since no lipase was inducedin the bacterial cells. However, no estolide production was observed(Heo et al. (2009) New Biotechnology 26, 105-108).

Hence, in summary there has previously been much study of enzymicconversion in respect of each individual step required to convert atriglyceride such as triolein to one or more fatty acid estolides—(i)lipase hydrolysis of triglyceride to produce one or more unsaturatedfatty acids (ii) hydroxylation of unsaturated fatty acid(s) by oleatehydratase and (iii) the esterification of hydroxy-fatty acid(s), againby lipase action. However, it has not previously been taught how tocouple use of a lipase with step (ii) so as to enable the conversion oftriolein or any other triglyceride containing oleic acid into10-hydroxystearic acid and esters thereof in a single one-pot reactionprocess.

SUMMARY OF THE INVENTION

As indicated above, it has now been found that by carrying out theabove-noted esterification step with a lipase in an aqueous bufferedsolution it is possible to integrate this step with both action of anoleate hydratase on an unsaturated fatty acid and lipase hydrolysis oftriglyceride to provide that fatty acid substrate in a single reactionmedium with high or total conversion of triglyceride and ofhydroxy-fatty acid to ester. This means carrying out the lipaseesterification step under very different conditions than currentlysuggested as preferable for such esterification such as use of animmobilized lipase in an apolar reaction medium.

Thus in one aspect, the present invention provides a method forproducing one or more esters of one or more hydroxy-fatty acids whereinat least one such hydroxy-fatty acid is a hydroxy-fatty acid obtainableby action of an oleate hydratase on an unsaturated fatty acid substratewith a cis C9-C10 double bond, said method comprising use of a lipasefor esterification of said at least one hydroxy-fatty acid in an aqueousbuffered reaction medium and under conditions which are compatible withproduction of said at least one hydroxy-fatty acid in situ by saidoleate hydratase.

It will be understood that such conditions do not preclude an increaseof temperature and/or pH variation after a period of oleate hydrataseactivity in the same reaction medium and whereby lipase use may occurunder conditions which are not optimal or even favourable for oleatehydratase activity. Where an oleate hydratase and lipase are providedtogether in the same aqueous reaction medium, then it may however bepreferred not to vary the temperature or pH but provide constant aqueousconditions for both activity of the OHase to produce said at least onehydroxy-FA in situ and esterification by the lipase as illustrated bythe exemplification (see Examples 1 to 4).

Thus, the reaction medium may be supplemented with said oleate hydratasein the presence of an appropriate unsaturated fatty acid substrate.Advantageously, the reaction medium may be further supplemented by atriglyceride which is hydrolysed by the same lipase as used for theesterification reaction. This provides in the reaction medium the fattyacid substrate for the OHase and thereby enables a one-pot enzymicprocess for obtaining one or more fatty acid estolides directly from thetriglyceride without need for any separation step.

The estolide product may be affected by the reaction conditions. Thus,while all the enzyme steps of such a one-pot enzymic method may becarried out at the same temperature, as indicated above, atwo-temperature method is not excluded from consideration in which thetemperature is firstly maintained at a temperature suitable for thechosen OHase, e.g. 30-35° C., and then increased to a highertemperature, e.g. to favour synthesis of higher estolide oligomers(longer estolides than dimers) with high conversion. Change of pH withtemperature is also not excluded from consideration to influence thefinal product(s) e.g. to permit lipase action at a pH not optimal forthe chosen OHase. However, such change of temperature and/or pH is notessential to the inventive concept which resides in enabling for thefirst time a one pot enzymic process for converting triglyceride toestolide product.

For example, as indicated above, such a process may provide one or morefatty acid estolides of 10-HSA starting from triolein as shown in thescheme set out in FIG. 3. In this case, the product will comprise,essentially consist of, or consist of one or more fatty acid estolidesselected from the monoestolide of 10-HSA with its parent unsaturatedfatty acid, i.e. oleic acid, the monoestolide of 10-HSA with itself, andhigher ester oligomers of 10-HSA capped or uncapped by oleic acid (seeFIG. 4). In some instances, it is conceivable that some cyclic ester mayoccur as a minor product. However, the lipase and conditions may bechosen so that exclusively open chain estolide(s) are obtained asillustrated by the examples herein.

A method of the invention as discussed above may further compriseextraction of the one or more ester products, e.g. a fatty acidestolide, from the reaction medium or such extraction and furtherpurification and/or incorporation into a composition. One or more esterproducts of lipase esterification may be reacted to obtain a furtherproduct either before or after extraction or after extraction andfurther purification.

BRIEF DESCRIPTION OF THE FIGURES

The invention will be further described below with reference to thefollowing figures:

FIG. 1: the hydration reaction of oleic acid catalysed by oleatehydratase to produce 10-HSA.

FIG. 2: the structures of examples of unsaturated C14-C18 fatty acidswith a cis double bond between C9 and C10 which are known to besubstrates for microbial oleate hydratases; monounsaturated fatty acids:myristioleic acid (C14; 9-cis-tetradecanoic acid), palmitoleic acid(C16; 9-cis-hexadecanoic acid), oleic acid (C18; 9-cis-octadecanoicacid); di- or tri-cis unsaturated fatty acids: linoleic acid (C18;cis-9,12-octadecanoic acid),α-linolenic acid (C18;(9Z,12Z,15Z)-octadecatrienoic acid) and γ-linolenic acid (C18; (6Z, 9Z,12Z)-octadecatrienoic acid); the hydroxyl-fatty acid ricinoleic acid(C18; 12(R)-hydroxy-9(Z)-octadecanoic acid).

FIG. 3: Scheme for the conversion of triolein to one or more fatty acidestolides by a one pot enzymatic process of the invention. The reactionconditions shown correspond to the reaction conditions illustrated bythe examples but may be varied provided the OHase and lipase both remainactive.

FIG. 4: Estolide formation in a one pot process of the invention wherethe monomers available for lipase esterification are oleic acid and10-HSA

FIG. 5: MALDI-TOF-MS spectral analysis of the ester products obtained bycarrying out a one-pot enzymatic process of the invention starting withtriolein; (top trace) the matrix; (middle trace) the solvent extractedester products obtained by the process of Example 1; (bottom trace) thesolvent extracted ester products obtained by the process of Example 2.

FIG. 6: MALDI-TOF-MS spectral analysis of the ester products obtained bycarrying out a one-pot enzymatic process of the invention starting withtriolein with 2 mg/ml oleate hydratase and Candida rugosa lipase at thefollowing concentrations: (A) 2 mg/ml; (B) 0.2 mg/ml; (C) 0.04 mg/ml(Example 3).

FIG. 7: MALDI-TOF-MS spectra of estolides obtained from 10-HSA withlipase from P. fluorescens in aqueous phase at 60° C. and pH 4, pH 6.5and pH 8, respectively.

FIG. 8: Diagram of the formation of magnetic chitosan compositeparticles for immobilization following glutaraldehyde activation ofrecombinant OHase with an N-terminal His tag.

FIG. 9: pH profiles of native and covalently immobilized OHase (seeExample 8): ▴ native OHase;

OHase-CHT-GL;

OHase-CMP;

OHase-AMP-CHT

FIG. 10: Thermal stability of native and immobilized OHase (see Example8): ▴ native OHase;

OHase-CHT-GL;

OHase-CMP;

OHase-AMP-CHT

FIG. 11: Variation of activity of OHase immobilized on magnetic chitosancomposite particles with multiple re-uses at 30° C. for 2 hours percycle with enzyme recovery by magnetic separation.

DETAILED DESCRIPTION OF THE INVENTION

As indicated above, the invention enables fatty acid estolide formationin an aqueous environment, at low temperature and ambient pressurewithout the need for separation and purification of intermediateproducts. It enables the integration of three consecutive reactionscatalysed by only two enzymes in a single reaction medium for derivingfatty ester estolides from triglycerides which are known to form a highpercentage of the lipid content of natural plant oils. It thus allowsthe production of high value compounds from bio-based feedstock withhigh efficiency and low energy cost.

Reaction Conditions

As noted above, the reaction conditions may be chosen to be fullycompatible with activity of an oleate hydratase to convert anunsaturated fatty acid substrate to the corresponding hydroxy-fatty acidin the same aqueous medium. In this case, generally the reaction mediumwill comprise an aqueous buffered solution at about pH 8.0, or at aboutpH 6-8 or at about pH 6 to 6.5. The optimum pH will be influenced by thechosen OHase and whether it is immobilized or not. In some instances,the OHase may show two distinct maximum activity values at about pH6-6.5 and about 8.0; in others the OHase may exhibit a single optimumactivity at about pH 8.0 or about pH 6-6.5 (see Example 8). For example,the reaction medium may preferably comprise a simple aqueous buffersolution at about pH 6.5, e.g. a simple phosphate buffer at pH 6.5. Insome instances, it may however be chosen to permit operation of thelipase at a different pH following a period of operation of the OHase.This will generally be within the range of about pH 4 to 8, for examplefollowing a period of operation of the OHase, e.g. 24 hours, the pH maybe lowered to about pH 6.5 or lower, e.g. about pH 4, to influence theproducts derived from the hydroxyl-FA(s) produced in situ by OHaseactivity.

The medium may preferably contain NaCl to improve the activity of theOHase, e.g. at a concentration up to about 150-200 mM, e.g. at about 150mM or at about 40 to 150 mM, about 40 to 100 mM, about 40 to 60 mM orabout 50 mM. The optimum concentration may again vary with the precisenature of the OHase employed. With the purified recombinant OHase of E.meningoseptica employed for the exemplification herein, NaCl at 150 mMwas found to be favourable (see Example 1).

As indicated above, a temperature may be chosen to be consistent withboth OHase activity and function of the lipase for esterification andhydrolysis of triglyceride. Thus a chosen single reaction temperaturemay be between about 10 and 50° C., preferably between about 20 and 40°C. Hence, the chosen temperature may generally be about 30-37° C. orabout 30-35° C. A temperature of about 30° C. may be chosen. Thus forexample an aqueous medium at about pH 6-8 and about 20-40° C. may beconveniently employed, e.g. at about pH 6.5 and about 30° C. asillustrated by the exemplification. However, if enzymes with a highthermal stability are used reaction temperatures above 50° C. may bepossible, e.g. a temperature of 60° C. or even 75° C. When enzymes withdifferent thermal stability are used, i.e. an oleate hydratase withlower thermal stability than that of the lipase, a step-increase of thetemperature may be used, i.e. starting at a lower temperature suitablefor OHase activity, e.g. 30° C. for a period of for example 24 hours,followed by increasing the temperature, e.g. to 60° C. for a period offor example another 24 hours.

The reaction medium may be additionally supplemented with one or moreadditives, e.g. to aid oleate hydratase activity in the reaction medium,e.g. FAD for an FAD-dependent OHase such as the OHase of Macrococcuscaseolyticus. A low amount of an organic solvent, generally lower than4-5% v/v, may be added to increase the solubility of the hydroxy-FA,e.g. 10-HSA. The solvent should not inhibit the enzymes and must bemiscible with the substrates (e.g. triolein and oleic acid) and theproducts.

One or more monomers may be included in the reaction medium foresterification which supplement the at least one hydroxy-FA derived froman oleate hydratase substrate and any such substrate remaining in thereaction medium. Such additional monomers may for example be non-fattyacid monomers capable of esterification by the lipase to the hydroxygroup of a hydroxy-FA. They may be a fatty acid monomer. One or moresaturated fatty acids may be added to cap estolide formation fromhydroxy-FA monomer(s). However, commonly the monomers available for thelipase esterification step will be restricted to the hydroxy-FAproduct(s) of oleate hydratase activity and the parent cis-9 unsaturatedfatty acid(s), e.g. 10-HSA and oleic acid, 10-hydroxyhexadecanoic acid(10-HHDA) and palmitoleic acid and 10-hydroxy-12-undecenoic acid(10-HUDA) and linoleic acid.

Where a triglyceride is added to the aqueous reaction medium for lipasehydrolysis, the two liquid phases will desirably be mixed thoroughly toform an emulsion. This may be achieved by spinning, e.g. at about 1000rpm, to aid dispersion of the triglyceride oil phase in the aqueousreaction medium.

The reaction may be stopped by addition of acid and the fatty acid esterproduct(s) extracted using an organic solvent e.g. dichloromethane. Thethus extracted product(s) may be analysed by MALDI-TOF-MS. In this way,the reaction may be monitored and the enzymes and/or conditions and/ortime of reaction varied to influence the ester product(s). It will beappreciated that the process of the invention is highly versatile andthe starting compounds and conditions may be varied to direct theprocess to the obtaining of a wide range of desired ester products.

The Ester Product(s)

The product or products of the lipase esterification step will berestricted by the available monomers for esterification. Generally, theesters will be one or more fatty acid estolides consisting solely offatty acid monomers. Thus as noted above, where a parentmono-hydroxy-FA, e.g. 10-HSA, is provided in situ by the action of anoleate hydratase, any of the following fatty acid estolides may beobtained: the monoestolide of the hydroxy-FA with its parent unsaturatedfatty acid, the monoestolide of the hydroxy-FA with itself, and higherester oligomers of the hydroxy-FA capped or uncapped by the parentunsaturated fatty acid.

Chemical hydroxylation of unsaturated fatty acids is difficult andfrequently di-hydroxy derivatives are obtained. In carrying out aprocess of the invention, it may be desirably ensured that solely one ormore mono-hydroxy-fatty acids are generated in situ for esterification.This will simplify the possible ester products. Thus in a preferredembodiment of the invention where only one or more mono-hydroxy fattyacids are provided in the reaction medium for lipase esterification, thepossible fatty estolide products may be restricted to one or more fattyacid estolides selected from a monoestolide of a hydroxy-FA with anunsaturated fatty acid, e.g. its parent cis-9 unsaturated fatty acid, amonoestolide of a hydroxy-FA with itself or another hydroxy-FA, andhigher ester oligomers formed from one or more hydroxy-FAs capped oruncapped by a non-hydroxy unsaturated fatty acid.

Thus if triolein is provided as the sole starting triglyceride for aprocess of the invention, solely oleic acid will be provided by lipasehydrolysis of the triolein as substrate for the oleate hydratase. Theoleate hydratase will in turn generate solely 10-HSA. As noted above,this restricts the possible open chain estolide products to themonoestolide of 10-HSA with oleic acid, the monoestolide of 10-HSA withitself, and higher ester oligomers of 10-HSA capped or uncapped by oleicacid. Where a single mono-10 hydroxy FA is provided for esterification,the lipase and conditions may be chosen to limit the estolide productsolely or essentially solely to monoestolide product.

Thus, the product distribution may be affected by the kinetics of eachreaction and factors including the choice of lipase and the ratio oflipase to OHase. For example, it may be arranged that the only, oressentially the only, ester product generated is monoestolide of anunsaturated fatty acid provided as substrate for the OHase and themono-hydroxy-FA resulting from action of the OHase on that substrate,e.g. the monoestolide of oleic acid with 10-HSA (see Example 1 using thelipase of Candida rugosa and FIG. 4). Alternatively, a process of theinvention with the same starting triglyceride may provide solely oressentially two different monoestolides: the monoestolide of amono-hydroxy-FA with itself, e.g. the monoestolide dimer formed from two10-HSA chains, and a monoestolide of that hydroxy-FA with its parentcis-9 unsaturated fatty acid (see Example 2 illustrating use of thelipase of a Pseudomonas species and FIG. 4).

It will be recognised however that a variety of estolide products may beachieved. For example, as will be expanded upon below, a broad range oflipases from different sources have been found to be able to catalyzethe synthesis of 10-HSA estolides and the choice of lipase and preciseconditions of lipase use can influence the nature of the estolidesattained. For example, in an aqueous system at pH 6.5 and at 40° C.,lipase from P. fluorescens has been found to synthesise mono- anddi-estolides of 10-HSA, with about 20% conversion, in 24 hours.Increasing the temperature to 60° C., polyestolides up to 15 mers havebeen found to be obtainable and conversion increases to 70%. Asindicated above, it is contemplated that such estolide formation mightbe combined with use of an OHase at lower temperature, e.g. at 30-35°C., to favour production of 10-HSA from oleic acid followed by a rise oftemperature to 60° C. for esterification. However, alternatively with anOHase of higher thermostability (due to immobilization and/or microbialsource) polyestolides higher than dimers may be achievable from a cis-9unsaturated fatty acid in a constant temperature process according tothe invention, e.g. operation at 50-60° C.

Choice of Lipase

Any lipase may be employed which will act at the secondary —OH group of10-HSA to form an ester bond. Such a lipase may be selected from themany lipases that have previously been shown to act at secondary —OHgroups of naturally occurring hydroxy-FAs such as ricinoleic acid andlesquerolic acid. Desirably the lipase may be a microbial lipase; it maybe a recombinant lipase.

Based on previous studies of lipases for esterification of hydroxy-FAs,generally the chosen lipase will be a non-1,3 positional specific lipasewith respect to triglyceride hydrolysis. It may be a non-specific lipasewith no positional specificity with respect to triglyceride hydrolysis.By way of example, non-specific lipases from the following fungal andbacterial species may be employed: Candida species, e.g. Candida rugosa,Candida antarctica, Candida lipolytica, Chromobacterium species, e.g.Chromobacterium viscosum, Geotrichum species, e.g. Geotrichum candidum,Pseudomonas species, e.g. Pseudomonas fluorescens, and Pseudomonasstutzeri, Alcaligenes species, Thermomyces species, e.g. Thermomyceslanuginosus; Thermoanaerobium brockii, Aspergillus oryzae, Rhizopusarrhizus and Mucor javanicus. For example, the lipases of C. rugosa, P.fluorescens and P. stutzeri have been shown by the inventors to besuitable, are commercially available and may therefore be convenientlyused.

The estolide synthesis can also be performed with immobilized lipasesthat can be easily removed from the reaction mixture and reutilised fora number of reaction cycles. Lipases immobilized using differentimmobilization methods such as adsorption, ionic binding, affinity,hydrophobic interaction, covalent binding, membrane encapsulation, gelentrapment and crosslinking are useful. Such lipases are known in theart and again are commercially available as noted above.

As indicated above, it is recognised that choice of lipase will be afactor which may influence the product distribution. For example, thelipase of Candida rugosa may be employed where the desire is to favourproduction of monoestolide between a hydroxy-FA, obtained or obtainableby action of an oleate hydratase, and its parent unsaturated FA (seeExample 1).

Choice of Oleate Hydratase

Where an oleate hydratase is employed to produce a hydroxy-FA in situ inthe reaction medium it will preferably be a recombinant oleatehydratase. As indicated above, the coding sequences for a number ofmicrobial oleate hydratase enzymes have previously been cloned and maybe readily identified. For example, it may be preferred to userecombinantly produced oleate hydratase of Elizabethkingiameningoseptica. As noted above, the coding sequence for that OHase haspreviously been deposited in GenBank and it may be readily expressed inE. coli, e.g. with a His-tag to aid purification.

Use of Immobilized OHase

As noted above, purified recombinant OHase is recognised to have lowstability even at ambient temperatures which limits its reuse incommercial applications. Immobilizing the enzyme is liable to cause lossof activity but may nevertheless be preferred to gain thermal stabilityand easy removal from the reaction mixture with re-cycling. Whilevarious methods for immobilization may be contemplated, studies of theinventors referred to above and now reported in more detail herein (seeExample 8) provide foundation for particular interest in recombinantOHase covalently linked to chitosan-coated magnetic particles. Suchcomposite particles in which smaller iron oxide magnetic particles, e.g.about 1 μm in diameter, are dispersed in a chitosan matrix may beconveniently prepared using commercially available magnetic particlessuch as amino-terminated iron oxide magnetic particles (see FIG. 8). Forthis purpose, the amino-terminated magnetic particles in water (e.g. at50 mg/ml) may be dispersed in an acidified chitosan solution, e.g. 2%(w/v) chitosan in 2% acetic acid, at, for example, a chitosan solution:wet magnetic particle ratio of about 5:1 (w/w). For immobilization ofrecombinant OHase with an N-terminal His tag, the composite magneticchitosan particles may be activated for covalent linkage of the OHaseusing glutaraldehyde.

Recombinant OHase of E. meningoseptica immobilized in this manner showedadequate recovered activity (higher than achieved with other recognizedenzyme immobilization methods; see Table 4 in Example 8) and importantlyas noted above, showed very effective improvement of operationalstability—the covalently bound enzyme preserved 75% of the initialactivity after five reuses at 30° C. for 2 hours per cycle (see Table 2in Example 8 and FIG. 11). This was accompanied by improved thermalstability. Such immobilized OHase can be readily recovered from areaction medium by magnetic separation and hence is now contemplated asfavourable for use in carrying out methods of the invention withrecycling of the OHase to new starting mixtures for batch wiseoperation.

Provision of Fatty Acid Substrate for an OHase

For a process of the invention, fatty acid substrate for an oleatehydratase may be added to the reaction medium as one or more cisunsaturated fatty acids, e.g. as one or more purified unsaturated cis-9unsaturated fatty acids or a triglyceride hydrolysate. However, asindicated above, more desirably fatty acid substrate for an OHase willbe generated in situ in the reaction medium by hydrolysis oftriglyceride by the same lipase as provided for the esterification step.

Thus as a preferred embodiment, the present invention provides a one-potenzymic method for producing one or more esters of one or morehydroxy-fatty acids wherein at least one such hydroxy-fatty acid is ahydroxy-fatty acid obtainable by action of an oleate hydratase on anunsaturated fatty acid substrate, said method comprising use of a oleatehydratase and a lipase in a single aqueous buffered reaction medium tocarry out the following steps without any separation step:

-   (i) hydrolysis of one or more triglycerides, e.g. triolein, by the    lipase to generate one or more unsaturated fatty acids as substrate    for said oleate hydratase;-   (ii) conversion of said one or more unsaturated fatty acids to one    or more hydroxy-fatty acids by said oleate hydratase; and-   (iii) conversion of said one or more hydroxy-fatty acids by the same    lipase to one or more esters.

As indicated above, the reaction mixture will generally be spun to aiddispersion of the triglyceride. Since unsaturated fatty acid, e.g. oleicacid, produced by lipase hydrolysis is immediately converted in the samereaction medium to hydroxy-FA, this favours full conversion oftriglyceride.

In the absence of additional monomers in the reaction medium capable ofester bond formation with the hydroxy-FA(s) produced in situ by theOHase, the ester products will be exclusively one or more fatty esterestolides. In the case of provision in the reaction medium of solely oneor more mono-10-hydroxy-FAs, it will be appreciated that the possibleestolides are any of a monoestolide of a hydroxy-FA with an unsaturatedfatty acid, e.g. its parent unsaturated fatty acid, a monoestolide of ahydroxy-FA with itself or another hydroxy-FA, and higher ester oligomersformed from one or more hydroxy-FAs capped or uncapped by a non-hydroxyunsaturated fatty acid.

The one or more unsaturated fatty acid substrates provided for theoleate hydratase may be any unsaturated fatty acid substrate that can beconverted by the hydratase to a hydroxy-FA. As indicated above, suchsubstrates are known to include not only oleic acid but otherunsaturated fatty acids with a cis double bond between C9-C10. Forexample, as indicated above, such substrates have been identified asC14-C18 unsaturated fatty acids with a cis-9 double bond, including anumber of such naturally-occurring fatty acids. Suitable unsaturatedfatty acids may have more than one cis double bond, e.g. at least both acis-9 and cis-12 double bond in which case as indicated above adihydroxy fatty acid may be produced with both a C10 and C13 hydroxygroup. Suitable unsaturated fatty acid substrates may have exclusivelycis double bonds. Thus suitable fatty acid substrates include forexample one or more of myristoleic acid, palmitoleic acid, oleic acid,linoleic acid, α-linolenic acid and γ-linolenic acid, all of which arefound as fatty acyl components of natural oils.

The OHase of E. meningoseptica (EC 4.2.1 53) is strictly specific for acis C9-C10 double bond. Thus, while other double bonds may be present, acis C9-C10 bond is considered required in any fatty acid substrate forthat enzyme. Thus, particularly if the recombinant OHase fromElizabethkingia meningoseptica is used for a one-pot process of theinvention, it is considered that a starting triglyceride must contain atleast one fatty acid chain with a cis double bond at C9-C10.

As indicated above, generally it will be preferred to provide foresterification only one or more mono-hydroxy-FAs, more preferably asingle 10-hydroxy-FA. Thus as indicated above, preferably an OHase inthe reaction medium may be solely contacted with oleic acid as its fattyacid substrate and used to provide solely 10-HSA for lipaseesterification.

Oleate hydratase and a lipase may, for example, be contacted withtriolein, or an oil-comprising triolein, in an aqueous buffered reactionmedium suitable for activity of both enzymes, e.g. an aqueous phosphatebuffer solution at pH 6.0-6.5 and 30-35° C. containing 50-150 mM NaCl,whereby the following steps occur consecutively without any separationstep:

-   (iv) hydrolysis of triolein to generate oleic acid;-   (v) conversion of oleic acid to 10-HSA by the oleate hydratase and-   (vi) conversion of 10-HSA to one or more estolides by esterification    with itself and/or oleic acid,

the reaction mixture desirably being continuously spun, e.g. at 1000 rpmfor 24-48 hours.

While triolein has a single type of fatty acyl chain, it will beunderstood that triglyceride(s) provided in the reaction medium forhydrolysis by the lipase may have a glycerol component esterified to asingle type of fatty acyl chain or more than one type of fatty acylchain, the only requirement being that such hydrolysis provides at leastone unsaturated fatty acid substrate for an oleate hydratase. One ormore purified triglycerides may be provided in the reaction medium suchas commercially available triolein. However, more desirably one or moretriglycerides may be provided as a component of a natural oil, oilywaste product or oil-containing preparation derived therefrom.

For example, for this purpose a natural plant oil may be employed. Ofparticular interest for this purpose are plant oils comprisingtriacylglycerols known to contain a high percentage of amono-unsaturated C18 fatty acid substrate for OHase, e.g. castor oil,corn oil, soybean oil, linseed oil, rapeseed oil, palm oil and sunfloweroil. A number of plant oils comprise a high content of triacylglycerolcontaining oleic acid and may therefore be of particular use where it isdesired to provide 10-HSA for estolide formation, e.g. olive oil, cornoil, linseed oil, canola oil, sunflower oil, soybean oil, peanut oil,pecan oil, macadamia oil, grape seed oil and sesame oil. The followingoils with a relatively high amount of triolein/oleic acid have beenchosen as a starting source of triglyceride by the inventors: olive oil(55-83% oleic acid), corn oil (20.0-42.2% oleic acid), linseed oil(14.0-40% oleic acid), sunflower oil (14-39.4% oleic acid), soybean oil(17-30% oleic acid), peanut oil (52-60% oleic acid) [Harwood et al. TheLipid Handbook with CD-ROM, 3rd edition, CRC Press 2007, pages 66-67].

Other natural oils of possible interest for biotransformation oftriglyceride to estolide in accordance with the invention includeoiticica oil and tall oil (a by-product of wood-pulp manufactureincluding resin acids and fatty acids including oleic acid). A startingtriglyceride source such as a natural oil or oily waste product mayprovide monomers for the final lipase esterification step in addition toone or more hydroxy-FAs via lipase hydrolysis and OHase action.Nevertheless, to simplify the end products for initial proof of conceptstudies, use of commercially available triolein was preferred asillustrated by the following examples.

EXAMPLES

The following examples 1 to 3 accord with the one-pot scheme shown inFIG. 3. Example 4 illustrates the same scheme in which triolein issubstituted by a natural oil containing a high percentage of the sametriglyceride.

Example 1: Synthesis of Monoestolide of 10-Hydroxystearic Acid withOleic Acid in a One-Pot Conversion of Triolein Using a Lipase from C.rugosa and Oleate Hydratase from Elizabethkingia meningoseptica

Enzymes

The lipase from Candida rugosa was obtained from Fluka. The hydrolyticactivity of the lipase was determined as 0.05 U/mg using p-nitro-phenylpalmitate as substrate. One unit will hydrolyse one micromole ofp-nitrophenol per minute from p-nitrophenol palmitate at 37° C. and pH8.

Recombinant OHase of E. meningoseptica with an N-terminal His tag wasobtained by using cell-free extract of E. coli TOP10 cells containingthe plasmid pBAD-HISA-OH (Bevers et al. (2009) J. Bacteriol. 191,5010-5012). The recombinant cells were grown at 37° C. in TB (TerrificBroth) medium supplemented with 100 μg ml⁻¹ ampicillin until the OD600reached the value 0.6-0.8. The expression of the recombinant enzyme wasinduced by using arabinose to a final concentration of 0.02%, followedby incubation at 30° C., 180 rpm overnight. Cells were harvested bycentrifugation (10,000 rpm, 30 min, 4° C.; Sorvall), washed with 20 mMTris-HCl pH 8, and lysed in the same buffer with a cell disruptor at 1.5kBar (Constant Systems, IUL Instruments). Cell free extract wasseparated from cell debris by centrifugation (4° C., 14,000 rpm, 30 min)and then filtered through a 0.45 μm filter. The enzyme was furtherpurified by Ni-affinity chromatography (His-tag purification) using aHisTrap-HP column (GE Healthcare), and an FPLC system equipped with UVdetection. The washing buffer during purification was 20 mM Tris-HCl, 50mM NaCl, 5 mM imidazole, pH 8.0 and protein elution was achieved with agradient up to 50% of the same buffer (20 mM Tris/HCl, 50 mM NaCl, 500mM imidazole, pH 8.0). Pooled fractions were concentrated and desaltedusing a Viva spin ultrafiltration tube (10 kDa cutoff; Vivascience), andconcentrated again to about 5-15 mg mL⁻¹ in 50 mM Tris-HCl buffer, pH8.0. Stock solutions of the enzyme were stored at −20° C. Purificationwas monitored by SDS-PAGE. Oleate hydratase was identified as a 70 kDaprotein band on SDS-PAGE. This is consistent with the known 646 aminoacids of the protein plus the hexa-histidine tag.

The activity of the OHase was determined by using oleic acid (2 mM) assubstrate in 500 μl 20 mM Tris-HCl, 150 mM NaCl, pH 8, by incubation at30° C., 1000 rpm for 2 hrs. The reaction was stopped by addition of 50μl HCl and the substrate and the product was recovered by threeconsecutive extractions with one volume of dichloromethane. Aftersolvent evaporation, the fatty acids were derivatized by usingbis(trimethylsilyl) trifluoroacetamide/trimethyl-chlorosilane (BSTFA),and analyzed by GC-MS. The determined OHase activity was 104.76 U/gprotein. One unit is the amount of enzyme converting one micromole ofoleic acid per minute, at 30° C. and at pH 8.

Earlier studies on the effect of NaCl concentration at 0 to 200 mM onthe activity of the OHase confirmed that NaCl was important foractivity. Activity was observed at all NaCl concentrations tested (50mM, 100 mM, 150 mM and 200 mM) but was optimal at 150 mM NaCl.Therefore, 150 mM NaCl was employed in all future reaction media forexemplification of the invention.

Reaction Process

Reaction mixtures containing lipase from Candida rugosa (10 mg/ml),oleate hydratase (0.2 mg/ml) and 10 mM triolein in 20 mM phosphatebuffer 150 mM NaCl, pH 6.5 were incubated at 30° C., 1000 rpm for 24hrs. The reaction was stopped by the addition of 50 μl HCl 2N and thesubstrate and products were extracted three times by using one volume ofdichloromethane. The solvent was evaporated at 40° C. and the productwas analyzed by GC-MS and MALDI-TOF MS.

MALDI-TOF-MS analysis identified the monoestolide of 10-hydroxystearicacid with oleic acid as the only product of the reaction, illustrated bythe peaks corresponding to the masses of molecular adducts [M-K]⁺, m/z:603.04 found 603.59 and [M-2K]⁺, m/z: 641.14 found 641.22 (see FIG. 5;middle trace). Glycerol was detected by GC-MS. Total conversion oftriolein was achieved, as well as total conversion of the 10-HSAobtained by OHase-catalyzed hydroxylation of the liberated oleic acid.

As a negative control, the same reaction was carried out using onlylipase from Candida rugosa (10 mg/ml) and without oleate hydratase. Theproduct of the control reaction contained only oleic acid produced fromtriolein by enzymatic hydrolysis. The product did not contain10-hydroxystearic acid or the monoestolide of 10-hydroxystearic acidwith oleic acid.

Example 2: Synthesis of Monoestolides of 10-Hydroxystearic Acid in aOne-Pot Conversion of Triolein Using a Lipase from P. fluorescens andOleate Hydratase from Elizabethkingia meningoseptica

Enzymes

Lipase of Pseudomonas fluorescens was obtained from Sigma-Aldrich, andhad a catalytic activity of 0.11 U/mg. One unit will hydrolyse onemicromole of p-nitrophenol per minute from p-nitrophenol palmitate, at37° C. and pH 8.

Recombinant oleate hydratase was employed as in Example 1.

Reaction Process

Reaction mixtures containing lipase from Pseudomonas fluorescens (5mg/ml), oleate hydratase (0.2 mg/ml) and 10 mM triolein in 20 mMphosphate buffer 150 mM NaCl, pH 6.5 were incubated at 30° C., 1000 rpmor 48 hrs. The reaction was stopped by the addition of 50 μl HCl 2N andthe substrate and products were extracted three times by using onevolume of dichloromethane. The solvent was evaporated at 40° C. and theextracted substrate and product were analyzed by GC-MS and MALDI-TOF MS.

MALDI-TOF-MS analysis identified the monoestolide of 10-hydroxystearicacid with oleic acid ([M-K]⁺, m/z: 603.04, found 603.31 and [M-2K]⁺,m/z: 641.14, found 641.49) and the monoestolide of 10-hydroxystearicacid with itself ([M-K]⁺, m/z: 621.04, found 621.54 and [M-2K]⁺, m/z:659.14, found 659.56), as reaction products (FIG. 5; bottom trace).Traces of triolein ([M-K]⁺, m/z: 923.53, found 923.76) and someunreacted 10-hydroxyoleic acid (([M-K]⁺, m/z: 376.68, found 377.22) werealso identified.

As a negative control, the same reaction was carried out using onlylipase from Pseudomonas fluorescens (5 mg/ml) and without oleatehydratase. The product of the control reaction contained only oleic acidproduced from triolein by enzymatic hydrolysis and a small amountunreacted triolein. The product did not contain 10-hydroxystearic acid,the monoestolide of 10-hydroxystearic acid with oleic acid or themonoestolide of 10-hydroxystearic acid with itself.

Example 3: Biotransformation of Triolein to Estolides at Different RatioLipase/Oleate Hydratase

Reaction mixtures containing triolein (14.3 mM, 12.4 mM and 13.2 mM) in20 mM phosphate buffer 150 mM NaCl, pH 6.5 were treated with oleatehydratase from Elizabethkingia meningoseptica (2 mg/ml) and variousamounts of lipase from Candida rugosa, e.g. 2 mg/ml, 0.2 mg/ml and 0.04mg/ml. The mixtures were incubated at 30° C., 1000 rpm for 24 hrs. Thereactions were stopped by the addition of 50 μl HCl 2N and the substrateand products were extracted three times by using one volume ofdichloromethane. The solvent was evaporated at 40° C. and the extractedsubstrate and product were analyzed by GC-MS and MALDI-TOF MS. Theresults are summarised in Table 1 and FIG. 6. As seen in Table 1, theratio between the activity of the lipase and the activity of oleatehydratase utilised has a large effect on the overall conversion oftriolein and the yield of estolide. No estolide was formed at very lowlipase/oleate hydratase ratio, although 58% of the oleic acid producedfrom triolein by hydrolysis was converted into 10-HSA.

TABLE 1 Results of the triolein conversion at different ratios betweenlipase from C. rugosa and oleate hydratase from E. meningoseptica (2mg/ml) Composition of product mixture at 24 h Total Oleic 10HSA con-acid conv. in version conv. 10HSA- Triolein Added oleic in 10 oleic acidcon- lipase Lipase/OHase acid HSA estolide version (mg/ml) weightactivity (%) (%) (%) (%) 2   1   0.48 81.0 56.8 42.4 >98 0.2  0.1  0.0579.2 50.7 36.4 90 0.04 0.02 0.01 58.0 58.0 0 32

Example 4: Biotransformation of Olive Oil to Estolides in a One-PotConversion Using a Lipase from C. rugosa and Oleate Hydratase fromElizabethkingia meningoseptica

Reaction mixtures containing 21.4 mg/ml of olive oil consisting ofapproximately 84% triolein in 20 mM phosphate buffer 150 mM NaCl, pH 6.5were treated with oleate hydratase from Elizabethkingia meningoseptica(2 mg/ml) and lipase from Candida rugosa (2 mg/ml). The mixture wasincubated at 30° C., 1000 rpm for 24 hrs. The reaction was stopped bythe addition of 50 μl HCl 2N and the substrate and products wereextracted three times by using one volume of dichloromethane. Thesolvent was evaporated at 40° C. and the extracted substrate and productwere analyzed by GC-MS and MALDI-TOF MS.

High conversion of the triolein component of olive oil was obtained.Oleic acid and 10-HSA were determined by GC analysis at a molar ratio of40:60, showing a high conversion of oleic acid under the action ofoleate hydratase. Formation of the monoestolide of 10-HSA capped witholeic acid was identified in MALDI-TOF-MS spectra. No products of thereactions (i.e. 10-HSA and estolides) were found by GC and MALDI-TOF-MSanalysis of control reactions without enzymes.

As indicated above, olive oil may be substitute in the protocol by otheroils with a high amount of triolein/oleic acid including sunflower oil,soybean oil, peanut oil, linseed oil and corn oil.

Example 5: Lipase Formation of Esters from 10-HSA

a) Screening for Lipases for Estolide Synthesis

Enzymes:

The following lipases were screened: Amano lipase from Pseudomonasfluorescens (P. fluorescens; 0.11 U/mg), Pseudomonas cepacia (P.cepacia; 0.37 U/mg), Thermomyces lanuginosus (T. lanuginosus; 1.09U/mg), and CLEA Alcalase were purchased from Sigma Aldrich. The lipasesfrom C. rugosa (0.05 U/mg), Aspergillus oryzae (A. oryzae; 1.30 U/mg),Candida antarctica A (C. antarctica A; 0.02 U/mg), Rhizopus arrhizus (R.arrhizus; 0.01 U/mg), Penicillium roqueforti (P. roqueforti; 0.01 U/mg),Mucor javanicus (M. javanicus; 0.16 U/mg), Candida lipolytica (C.lipolytica; 2.59 U/mg), Thermanaerobium brockii (T. brockii; 1.31 U/mg)were obtained from Fluka. Novozyme 435, Lipozyme TL, Candida antarcticaB (C. antarctica B) lipase were purchased from Novozymes. The lipasesfrom Alcaligenes sp (lipase TL; 3.86 U/mg), Pseudomonas stutzeri (P.stutzeri; 1.21 U/mg) (Lipase TL), were products of Meito Sangyo Co. Ltd,Japan. The CLEA from C. antarctica B and P. stutzeri were purchased fromCLEA Technologies. One unit will hydrolyse one micromole ofp-nitrophenol per minute from p-nitrophenol palmitate, at 37° C. and pH8.

Process:

The reactions were performed by adding native and immobilized lipases,50 U/mmol substrate, to a 1 mM solution of 10-hydrostearic acid broughtto a final volume of 1 ml in toluene, at 60° C. at 350 rpm for 24 h. Atthe end of the reaction the enzyme was removed by centrifugation (13,000rpm, 3 min), and the reaction mixture was analyzed by GC-MS andMALDI-TOF MS.

The reaction product was derivatized with BSTFA+TMCS (99:1), at 2:1reagent: sample ratio (w/w), for 1 h at 95° C., and analyzed by GC-MS,using hexadecane as internal standard. For the calculation ofconversions, the concentration of each substrate was determined based ona calibration curve in the range from 0.05 mM to 3 mM, using hexadecaneas internal standard. For the MALDI-TOF MS analysis, samples were mixedwithtrans-2-[3-(4-t-butyl-phenyl)-2-methyl-2-propenylidene]malononitrile(DCTB) matrix and potassium trifluoroacetate (KTFA) as ionization agent.10 μL of the sample was mixed with 10 μL of matrix solution (40 mg/mLDCTB solubilized in THF) and 3 μL of KTFA (5 mg/mL). About 0.3 μL of themixture was applied on the plate and measured in the positive mode.

The results given in Table 2 show that all lipases tested, except thelipase from P. cepacia, are able to convert 10-HSA into thecorresponding monoestolide (ME). Lipases from A. oryzae, C. antarcticaA, C. rugosa and P. fluorescens produced small amounts of cyclic diester(CDE) in low amounts, ranging from 1.2% (A. oryzae) to 10% (P.fluorescens). The lipase from P. stutzeri, both free and immobilized,produced low amounts of longer estolides, e.g. diestolides (DE) andtriestolides (TE). Highest 10-HSA conversion was obtained withimmobilized enzymes, most probably due to the increased stability.

TABLE 2 Synthesis of estolides from 10HSA using free and immobilizedlipases 10HSA Product type Lipase conversion (MALDI) Free enzymesAlcaligenes PL 52.5 ME, CDE A. oryzae 21.4 ME, CDE C. antarctica A 15.9ME, CDE C. antarctica B 21.4 ME C. lipolytica 21.5 ME C. rugosa 18.4 ME,CDE M. javanicus 12.9 ME P. cepacia  0.0 Not found P. fluorescens 25.3ME, CDE P. stutzeri 34.4 ME, DE P. roqueforti  3.1 ME R. arrhizus  9.6ME T. brockii 31.9 ME T. lanuginosus  9.2 ME Immobilized enzymesLipozyme 57.3 ME Novozym 435 65.7 ME CLEA CalB 65.1 ME CLEA P. stutzeri66.5 ME, DE, TE CLEA Alcalase 35.3 ME

b) Synthesis of Estolides by Lipase-Catalyzed Conversion of10-Hydroxystearate in Aqueous Phase at Different Temperatures

Enzymes: Lipase from Pseudomonas fluorescens (P. fluorescens; 0.11 U/mg,obtained from Sigma) and lipase from Pseudomonas stutzeri (P. stutzeri;1.21 U/mg, obtained from Meito Sangyo Co. Ltd, Japan) were used in theseexperiments. One unit will hydrolyse one micromole of p-nitrophenol perminute from p-nitrophenol palmitate, at 37° C. and pH 8.

Reaction: To a suspension of 15 mM 10-hydroxystearic acid in 20 mMphosphate buffer of at a given pH, e.g. pH 4, pH 6.5 or pH 8, 100 μl ofenzyme solution were added, to an enzyme concentration of 50 U/mmolsubstrate. The total volume of the reaction mixture was 1 ml. Thereaction mixture was stirred at 350 rpm for 24 h. Reactions were carriedout at 40° C. and 60° C. respectively. As a negative control, the samereaction was carried out at identical conditions but without theaddition of the enzyme. At the end of the reaction the enzyme wasremoved by centrifugation (13,000 rpm, 3 min), and the reaction mixturewas analyzed by GC-MS and MALDI-TOF MS.

Samples were treated prior to analysis as described in Example 5a. FIG.7 gives the MALDI-TOF-MS spectra of the products produced at acidic pHvalues. The MALDI-TOF-MS spectra of control reactions contained only themass corresponding to the substrate 10-HSA ([M-K⁺], m/z: 377.04). Theresults in Table 3 show the increase of 10-HSA conversion into estolidesas well as the formation of longers estolides [the estolides ranged frommonoestolides (i.e. dimers) to E14 (i.e. 15-mers)] with increasingtemperature from 40 to 60° C. at acidic pH values, e.g. pH 4 and pH 6.5.Low temperatures, e.g. 40° C. and pH 6.5 are favourable for theproduction of monoestolides of 10-HSA as a sole product.

TABLE 3 Conversion of 10-HSA with lipase from P. fluorescens and P.stutzeri in buffer of different pH values, at 40 and 60° C. 40° C. 60°C. 10HSA Product 10HSA Product conv. Mw/ conv. (%) Mw/ pH (%) type PDItype PDI PD P. fluorescens lipase 4 28 ME (82%), 715/ 70 1639/ 2-15 DE(18%) 1.03 1.16 6.5 18 ME (62%), 733/ 68 1713/ 2-9  DE (32%) 1.03 1.15 85 ME 633/1 31 1514/ 2-9  1.15 P. stutzeri lipase 4 0 0 0 25 1645/ 2-121.18 6.5 0 0 0 43 1187/ 2-7  1.14 8 0 0 0 8 853/ 2-4  1.07 ME:monoestolide, DE: diestolide, TE: triestolide; Mw: average molecularweight (g/mol), PDI: polydispersity index; PD: degree of polylmerisation

Example 7: Hydration of Oleic Acid with Oleate Hydratase from E.meningoseptica

10-hydroxystearic acid was obtained by hydration of oleic acid usingoleate hydratase from E. meningoseptica. 5 ml of cell free extract ofOHase expressed in E. coli TOP10 cells (48 mg/ml total protein content)were added to an emulsion containing 0.6% (v/v) oleic acid in 20 mMTris-HCl buffer, pH 8.0 and the mixture was incubated at 30° C. at 200rpm for 12 h (total volume 50 ml). The reaction was terminated byaddition of 100 μl of 4 N HCl 4 N (final pH 1-2), and the product 10-HSAthat separates from the mixture as a white precipitate, was isolated byfiltration, dissolved in acetone and isolated after solvent removalunder vacuum (475 mg, 1.55 mmol, 10HSA yield 96%, purity >99%, asdetermined by GC, GC-MS and NMR).

Example 8: Increasing Operational and Thermal Stability of OleateHydratase by Immobilization

Recombinant oleate hydratase (OHase) of Elizabethkingia meningoseptica,expressed in E. coli and purified as described above, was immobilized bydifferent immobilization strategies including adsorption, crosslinking,entrapment and covalent bonding. Among the tested immobilizationmethods, covalent binding onto magnetic chitosan composite particles wasmost efficient; immobilization yields higher than 90% and recoveredactivities of up to 24% were achieved by covalent binding onto suchcomposite particles. This is a good result for such a difficult andunstable enzyme. The resulting biocatalysts were further characterizedin detail in terms of stability and reusability. The thermal stabilitywas enhanced after immobilization. The OHase immobilized on magneticchitosan composite particles retained more than 65% of its originalactivity after incubation at 50° C. for 2 hours, while the native enzymewas completely inactivated. Importantly, it was also surprisingly foundthat immobilization of the OHase on magnetic chitosan compositeparticles resulted in a radical improvement of operational stability ofthe OHase, as the covalently bound enzyme preserved 75% of the initialactivity after five reuses. This renders such immobilized OHase ofparticular interest for commercial application of one-pot methods of theinvention.

As noted above, an important concern for using isolated OHase forindustrial purposes has been its low stability, even at ambienttemperatures. As no straightforward approach for establishing a goodimmobilization method for a specified enzyme is known, this has to bedetermined experimentally by trial (Liese & Hilterhaus (2013), Chem.Soc. Rev. 42, 6236-6249).

Immobilization Methods

Chemicals

Oleic acid 96%, chitosan 85% deacetylated, amino-terminated magneticparticles (AMP), glutaraldehyde 50%, carboxyl terminated magneticparticles (CMP), fluorescein isothiocyanate (FITC), Span 80 (sorbitaneoleate), bis(trimethylsilyl)trifluoroacetamide/trimethyl-chlorosilane(BSTFA+TMCS=99:1) were purchased from Sigma Aldrich. The standard gradeSepabeads EC-EP were kindly provided by Resindion S.R.L. (Italy), Ni-NTASuperflow resin was purchased from Qiagen, Celite 545 was obtained fromMerck. Silane precursors dimethyldimethoxysilane (DMeDMeOS, 96%),tetraethoxysilane (TEOS, 98%) were purchased from Fluka Chemie GmbH(Buchs, Switzerland). Isobutyl trimethoxysilane (iBuTMOS, 97%) and(3-aminopropyl)trimethoxysilane (3-NH₂PrTMOS, 97%) were obtained fromSigma Aldrich (Steinheim, Germany), while tetramethoxysilane (TMOS, 99%)was obtained from Acros Organics (Geel, Belgium).

Activity Assay

Activities for the free and immobilized OHase were determined by usingoleic acid (2 mM) as substrate in 500 μl 20 mM Tris buffer, 150 mM NaCl,pH 8, by incubation at 30° C., 1000 rpm for 2 hrs. The reactions werestopped by addition of 50 μl HCl 3N and the substrate and the productwere recovered by three consecutive extractions with one volume ofdichloromethane. After solvent evaporation, the fatty acids werederivatized by using BSTFA (Hudson et al. (1995) Appl. Microbiol.Biotechnol. 44, 1-6) and analyzed by GC-MS. The separation was carriedout on Interscience Trace GC Ultra GC+PTV with AS3000 II autosamplerequipped with Restek Rxi-5 ms 30 m×0.25 mm×0.25 μm capillary column,using the following conditions: oven temperature: 100-300° C. with 10°C. min-1 heating rate, injector temperature 300° C., carrier gas(helium) flow 1.0 mL min-1. Hexadecane was used as internal standard.Mass spectra were obtained from an Interscience Trace DSQ II XLquadrupole mass selective detector (El, mass range 35-500 Dalton, 150 mssampling speed), mass spectrometer operated at 70 eV.

The specific activity was expressed as the amount of 10-HSA (μmol)formed by 1 g of enzyme protein in 1 min.

The recovered activity was defined as % ratio of the total activity ofthe immobilized enzyme and the total activity of the native enzyme usedfor immobilization.

Immobilization Method 1: Absorption

20 mg Celite 545 were mixed with 500 μL of OHase solution (2.87 mg ml-1)in 20 mM phosphate buffer, pH 8. The mixture was shaken at 4° C. for 48h. The adsorbed Celite-OHase was separated by centrifugation and washedfive times with 20 mM phosphate buffer pH 8 and two times with 20 mMTris buffer. The protein concentration was determined in each washingstep (except the washes with Tris buffer) by using the Bradford assay.The Celite-OHase was stored in 20 mM TRIS buffer at 4° C. until furtheruse. The immobilization in the presence of an emulsifier was carried outby the same procedure, adding 30 μl emulsifier (Span 80) in theimmobilization solution.

Method 2: Ni-NTA Agarose Beads Immobilization

1 ml of OHase solution (2.5 mg ml-1) in 20 mM Tris buffer pH 8 wereadded to 200 μl Ni-NTA Superflow resin. The mixture was shaken at roomtemperature for 1.5 h, 100 μm. The resin was washed three times withTris buffer and stored at 4° C. until further use.

Method 3: Ionic Binding on Chitosan

500 μl of 20 mM phosphate buffer, pH 6.5, were added to 25 mg chitosan,and the suspension was shaken at room temperature for 3 h, 100 rpm. Thewet chitosan was separated from buffer solution by centrifugation at14,000 rpm for 10 min, 500 μl OHase solution in 20 mM phosphate bufferpH 6.5 (2.5 mg ml-1) were added and the mixture was shaken at 4° C., 100rpm for 24 hrs. The washing step was performed as described for method1.

Covalent Binding onto Different Supports

Sequence analysis of OHase reveals a high density of 50 Lys groups whichcan be involved in covalent binding with various activated supports.

Method 4a: Epoxy-Sepabeads

25° C./4° C. 900 μl OHase solution (1.15 mg ml-1) in 50 mM phosphatebuffer pH 8 were added to 100 mg immobilization support activated withepoxy groups. The mixture was incubated overnight at 100 rpm, 24 h, at4° C. and 25° C., respectively. A set of experiments were performed bytreating with 3M glycine solution in 20 mM phosphate buffer pH 6.5,overnight at 4° C., 100 rpm, in order to block all free epoxy groups.The epoxy-sepabeads were removed from the mixture by filtration, washedas described for method 1 and stored at 4° C. until further use.

Method 4b: 4° C.-Three Steps

OHase immobilization in three steps was performed as previouslydescribed by Mateo et al. (2002) Biotechnol. Prog. 18 629-634 at pH 7 in1 M sodium phosphate for 24 hrs, followed by incubation of theimmobilized enzyme at pH 9 in 100 mM sodium phosphate for 72 h andhydrophilization of the support surface by incubating the derivative for24 hrs at pH 8.5 in the presence of 3 M glycine.

Functionalized Magnetic Particles (Amino Terminated, CarboxylTerminated)

Method 5a: Amino-Terminated Magnetic Particles (AMP)

200 μl AMP suspension, containing magnetic iron oxide particlesapproximately 1 μm in size, were activated three times by using couplingbuffer (0.01 M pyridine in distilled water pH 6, adjusted with 2N HCl)to a final volume of 1 ml and vigorous shaking. The supernatant wasaspirated and 400 μl of 5% glutaraldehyde solution were added. Theparticles were re-suspended and gently shaken at room temperature for 3hrs. The supernatant was aspirated and the wet cake was washed threetimes in order to eliminate the unreacted glutaraldehyde. 300 μl ofOHase solution (5.74 mg ml-1 in 20 mM phosphate buffer, pH 8) were addedto the glutaraldehyde-activated particles and after re-suspension of theparticles the mixture was gently shaken at 4° C. for 48 hrs. Theparticles were then separated magnetically and re-suspended in 500 μl ofGlycine Quenching solution (1.0 M in distilled water pH 8, adjusted with2N NaOH), for 30 min at room temperature, 100 rpm. At the end a washingstep was performed three times by washing buffer (0.01 M Tris basecontaining 0.15 M NaCl, 0.1% (w/v) bovine serum albumin, 0.001 M EDTAsodium salt and 0.1% (w/v) sodium azide), and three times by 20 mM Trisbuffer, pH 8. The immobilized enzyme was stored at 4° C. in 20 mM Trisbuffer, pH 8.

Method 6: Carboxyl-Terminated Magnetic Particles (CMP)

250 μl CMP suspension were activated 3 times by using coupling buffer(0.01 M phosphate buffer with 150 mM NaCl in distilled water, pH 5.5,adjusted with 2N HCl) to a final volume of 0.5 ml and shakingvigorously. The particles were re-suspended in 250 μl coupling buffer,100 μl coupling agent (1-ethyl-3(3-dimethylaminopropyl) carbodiimideEDCl, ˜0.6 mg ml-1), 250 μl OHase solution in phosphate buffer (5.74 mgml-1) were added, and the mixture was gently shaken at 4° C. for 48 hrs.Washing and storing conditions were the same as with AMP (see above).

Method 7: Covalent Binding on Pre-Activated Chitosan (CHT-GL)

400 μl glutaraldehyde 25% were added to 25 mg chitosan in 1.6 ml 20 mMphosphate buffer, pH 6.5. The suspension was shaken at room temperaturefor 3 h, 100 rpm. The glutaraldehyde activated chitosan was washed threetimes with 2 ml 20 mM phosphate buffer pH 7. 500 μl OHase solution (2.5mg ml-1) was added and the mixture was shaken at 4° C., 100 rpm, for 24hrs. The washing step was performed as described for method 1.

Method 8: Covalent Binding on Magnetic Chitosan Composite Particles(AMP-CHT).

Magnetic chitosan composite particles, in which amino-terminatedmagnetic particles (AMP) as described above are dispersed in a chitosanmatrix (see FIG. 8), were prepared as described by Kumar et al. (2013)Biotechnol. Bioprocess Eng. 787-795) with some modifications. Chitosan2% (w/v) solution in acetic acid (2%) was added to 187.5 mg wet AMP in5:1 ratio (w/w). The mixture was vigorously shaken and kept 1 hr in asonication bath for complete homogenization. The macroparticles wereprecipitated into sodium hydroxide 1M solution containing 26% ethanol asdescribed elsewhere [Biró et al. (2008) J. Biochem. Biophys. Methods 70,1240-1246), washed with distilled water and stored at 4° C. untilfurther use. Before immobilization, the particles were activated byadding 5% glutaraldehyde for 4 hrs at 10° C. in 20 mM phosphate bufferof pH 6.5. 250 μl OHase solution (2.5 mg ml-1) were added to 150 mg wetmacroparticles, completed to a total volume of 1 ml with 20 mM phosphatebuffer pH 7, and gently mixed overnight at 10° C. The immobilized enzymewas washed as previously described for method 1, and stored at 4° C. in20 mM Tris buffer pH 8.

Method 9: Cross-Linked Enzyme Aggregates (CLEA)

250 μl OHase solution (2.3 mg ml-1) was mixed with 750 μl saturatedammonium sulfate solution, adjusted to pH 8 with NaOH. The mixture wasshaken at 500 rpm at 4° C. for 112.5 μl (0.3%, w/v) of 25% (w/v)glutaraldehyde was added drop wise into the tube and the mixture wasshaken at 4° C., 500 rpm for 3 h. The CLEAs were removed bycentrifugation (14,000 rpm, 30 min), washed three times with 20 mMphosphate buffer pH 8 and two times with 20 mM TRIS buffer, and storedin the same buffer until further use.

Method 10: Sol-Gel Entrapment

The OHase sol-gel entrapment has been performed mainly as the previouslydescribed method for subtilisin (Corici et al. (2011) J. Mol. Catal. B:Enzym. 73, 90-97). In a 4 ml glass vial OHase solution (195 μl,containing 5.87 mgml-1, in 20 mM Tris buffer pH 8), 1M NaF (25 μl), 4%PEG 20,000 solution (25 μl) and isopropyl alcohol (50 μl) were mixed(magnetic stirring, 600 rpm). 1.5 mmol of silane precursors in differentmolar ratio were added and the mixing continued at room temperatureuntil the gelation started. The gel was kept 24 hrs at 4° C. to completepolymerization and then the bulk gel was washed to eliminate unreactedmonomers and additives with Milli-Q water (2.5 ml), isopropyl alcohol(1.25 ml) and n-hexane (1.25 ml) and dried at 25° C. for 24 hrs. Thesol-gel encapsulated enzyme was crushed in a mortar and stored at 4° C.until further use.

Influence of pH on OHase Activity

The effect of pH on native and immobilized OHase activity was evaluatedin the pH range 4-9 in 9 steps, by using a broad range of pH buffersolutions containing citric acid, boric acid and trisodium phosphatebuffer.

Influence of Temperature on OHase Activity

Incubation of the native/immobilized enzyme was carried out for 2 hrs inthe absence of the substrate at different temperatures, in the 30-50° C.range, followed by cooling on ice for 10 min. The residual activity ofthe enzyme was then determined.

Reuse of the Biocatalyst

The activity of immobilized OHase was determined after repeated use ofthe biocatalyst at 30° C. for 2 hours per cycle. After each cycle, theimmobilized enzyme was separated and washed with 20 mM Tris Buffer, 150mM NaCl, pH 8, for 3 times. Fresh buffer and substrate were added, theactivity of the reused biocatalyst was assayed and the obtained valuewas compared to the first run (defined as 100%).

Morphological Characterization of Immobilized OHase

The OHase protein was labelled with FITC (based on PIERCE EZ-Label™ FITCLabeling Kit). The coupling reaction of OHase with FITC was started byadding dropwise 600 μl of FITC (1 mg ml-1 dissolved indimethylformamide) into the OHase solution (5.87 mg ml-1 in phosphatebuffer pH 8). The mixture was incubated for 1 h at room temperature. Thelabelled OHase was separated from unreacted FITC by several washes withphosphate buffer 20 mM pH 8, using a centrifugal filter device(Centricon PL-30, with a membrane nominal molecular weight limit of30,000 Da). UV-VIS spectra were collected after each washing step, untilthe absorbance at 493 nm (characteristic values of FITC) decreased up to0.1 absorbance units. The protein concentration was determined by theBradford assay. The FITC labelled protein was immobilized based on themethod described for covalent binding on magnetic chitosan compositeparticles and the fluorescence micrographs were registered by a LeicaTrue Confocal Scanner (Leica TCS SPE), with 10 fold spot magnitude.

Results and Discussion

The results are presented in Table 4 and are discussed in detail foreach immobilization method in this section.

Adsorption:

Immobilization of OHase on Celite 545, a slightly hydrophobic support,resulted in low protein loading and low recovered activity. (Table 4,entry 1). The lack of activity could be attributed to the hydrophilicregions on the enzyme surface that lead to desorption in aqueousenvironment, surpassing the interaction between the hydrophobic part ofthe enzyme surface and the hydrophobic support. In fact, most of theenzymatic protein was recovered in the washing steps. By using Span 80as additive during the immobilization process, the protein loadingincreased up to 3 times showing that the adsorption of the enzyme wasmore effective in the presence of an emulsifier, but the activityremained low.

Ionic Binding:

The ionic binding of OHase onto chitosan, an amino-functionalpolysaccharide with the pKa of the amino group of 6.5 was performed atthis pH (i.e. 6.5) since the net charge of the enzyme calculated basedon the amino acid sequence, is about −12.0. The protein loading value(Table 4, entry 4) exceeded 95%, but the specific activity of 11.8 U/gprotein corresponds to only 5.5% recovered activity.

Affinity:

Because the recombinant OHase had a His-tag and Ni-affinitychromatography was used to purify the enzyme with high protein recovery,this method was also studied as an immobilization tool. The Ni-Superflowresin led to total binding of the enzyme, but unfortunately theimmobilized enzyme showed very low specific activity and less than 5% ofthe initial activity was recovered (Table 4, entry 3). The resultssuggest that immobilization of His-tag enzymes by affinity is notuniversally applicable, although the method has been successfully usedfor immobilization of PikC hydroxylase (bacterial P450), p-nitrobenzylesterase, benzaldehyde lyase and horseradish peroxidase.

Cross-Linked Enzyme Aggregates:

OHase CLEA obtained showed high specific activity compared with thephysical immobilized enzyme (Table 4, entry 5), but the recoveredactivity did not exceed 17%.

Entrapment in Silica Sol-Gel Matrices:

This approach has been previously applied for the sol-gel entrapment oflipases designated to work in organic solvents as well as forisubtilisin and nitrile hydratase, which exhibited high enough. In thepresent study, the OHase was fully entrapped in all sol-gel preparations(Table 4, entries 12-15), but it did not show catalytic activity.

Covalent Binding:

The recovered activities (Table 4, entries 6-11) spanned over a widerange, between 1.0% and 23.7%, even if the immobilization protocols wereperformed under mild conditions. The covalent binding on epoxy sepabeadswas performed at different temperatures in one or three steps, in orderto avoid the loss of enzyme activity during the immobilization process.However, the use of epoxy-sepabeads as carrier for OHase (Table 4, entry9 and 10) yielded less than 5% recovered activity. The recoveredactivity of OHase immobilized onto sepabeads was low, even when glycinewas first used to block a part of the available reactive epoxy groups.Therefore, the drop in activity cannot be attributed solely tomultipoint covalent attachment.

The results obtained when chitosan was used as support for OHase showthe suitability of this natural support to accommodate the enzyme. Seespecific activity of 27.2 U/g, (Table 4, entry 8).

Covalent Binding of OHase on to Functionalized Magnetic Particles:

Recovered activities up to 20% corresponding to 42.5 U/g were obtainedwhen carboxyl terminated magnetic particles were used as support, butsome operational difficulties occurred during the magnetic separation ofthe immobilized enzyme, owing to the heterogeneity of the reactionsystem. The 10HSA product is a white solid and partial adsorption ofthis compound, as well as of the unreacted substrate on the surface ofthe magnetic particles could not be avoided. For this reason, modifiedmagnetic particles, coated with a few atomic layers of chemically activepolymer to provide functional groups for linkage were also studied.Chitosan was investigated as a suitable layer for magnetic particles onthe basis of the experiments with chitosan noted above.

Magnetic Chitosan Composite Particles (AMP-CHT):

Using such particles as support and glutaraldehyde as linker, about 24%(about 51 U/g) of the initial activity was recovered following theimmobilization. As noted above, this was considered a good result. Thehigher immobilization efficiency compared to other methods is due to theavailability of numerous lysine groups near to the surface for covalentbinding. The explanation of the partial loss of activity compared to thenative enzyme could be the intramolecular linking of glutaraldehydebetween lysine residues of the enzyme, or the possible mismatchedorientation of the enzyme on the carrier caused by the presence of a Lysresidue close to the entrance to the active site.

TABLE 4 Immobilization yield and recovered activity of OHase immobilizedby different methods. Protein Recovered Specific Immobilization Entryloading activity activity Method No. Type of solid support [%] [%] [U gprotein⁻¹] Adsorption  1 Celite 545 32.0 0 0  2 Celite 545 + Span 83.02.9 6.2 Affinity  3 Ni-NTA Superflow resin 99.3 1.8 3.9 Ionic binding  4CHT 97.9 5.5 11.8 Cross-linking  5 — 99.8 16.5 35.4 Covalent  6 AMP 97.6~2.0 4.3 binding  7 CMP 98.4 19.8 42.5  8 CHT-GL 99.2 12.7 27.2  9Epoxy-SB: 25° C./4° C. 99.5 <1.0 2.1 10 Epoxy-SB: 4° C. - three steps99.6 ~2.0 4.2 11 AMP-CHT 99.3 23.7 50.8 Sol-gel 12 TEOS/TMOS/TEOS:TMOS =1 98.6 0 0 entrapment 13 TEOS:iBuTMOS = 1:1 98.4 0 0 14 TEOS:DMeDMeOS =1:1 98.7 0 0 15 TEOS:3NH₂PrTMOS = 1:1 ÷ 1: 98.1 0 0

Effect of pH on the Catalytic Activity

The effect of pH on the activity of native and immobilized enzyme wasstudied in the pH range 4.0-9.0 using the three most active covalentlyimmobilized OHase preparations (AMP-CHT, CMP and CHT-GL); see FIG. 9.Two distinct maximum activity values for the native enzyme were found atpH 6.5 and 8.0. The same behaviour was observed using chitosan as theimmobilization support. Such behaviour is usually due to the existenceof two active isoenzymes that slightly differ in their structure, butthis is not the case for OHase. An important modification of the pHprofile occurred when both forms of functionalized magnetic particleswere used as the immobilization support. These two preparations wereactive in a larger pH range compared to the native enzyme and their pHprofile showed only one activity optimum, at pH 6.5 and pH 8.0 for OHaseimmobilized on CMP and AMP-CH, respectively. The most reasonableexplanation of the occurrence of multiple peaks in the pH profile ofOHase is the different physical state of the substrate, oleic acid,depending on the pH of the solution. At pH values less than 7, oleicacid is in the oil phase and the carboxylic groups are protonated. AbovepH 7, an increase of ionization occurs and more structured lamellarsystems or large vesicles of oleic acid are formed. It seems that boththese forms are suitable to interact with the enzyme active site. Thedrop in the activity of the native enzyme at pH 7 is probably due to thelower reactivity of the non-ionized form of the substrate. In case ofthe enzyme immobilized on functionalized magnetic particles,interactions with the free carboxyl or amino groups still existing onthe surface of the particles probably led to a certain extent ofionization even at pH 7 and increased the accessibility of the substratefor the hydration reaction.

Thermal Stability

The thermal stability of the same OHase preparations was investigated;see FIG. 10. All immobilized OHases displayed better thermal stabilitycompared to the native enzyme demonstrated by the higher values ofactivity at temperatures above 40° C. The highest relative activityvalue was achieved when AMP-CHT was used as support. The higher thermalstability can be attributed to the restricted flexibility of the enzymeafter immobilization, rendering it more resistant to unfolding anddenaturation. In the batch wise reaction, as noted above, the OHaseimmobilized on magnetic chitosan composite macroparticles retained morethan 65% of its original activity after incubation at 50° C., while thenative enzyme was completely inactivated. Such a significant improvementmakes this enzyme available for utilization in a temperature rangeconsidered normal for biocatalytic processes, at sufficiently highreaction rates.

Repeated use of the immobilized OHase

OHase immobilized on magnetic chitosan composite particles was evaluatedthrough several repetitive uses. After each cycle, the biocatalyst wasrecovered by magnetic separation, washed and recycled for 10-HSAproduction from oleic acid. Following acidification of the supernatantsolution, the product and the unreacted raw material were extractedthree times by dichloromethane. The activity of the first batch has beentaken as reference. FIG. 11 shows the variation of immobilized OHaseactivity during multiple reuses by magnetic separation. As noted above,after five reaction cycles, the relative activity still remained about75%, an excellent value taking into account the poor stability of theenzyme in the native form. Furthermore, in thinking about practicalapplication, the reuse of the enzyme is considered to fully counteractthe loss of activity during the immobilization.

Morphological Characterization of AMP-CHT Particles with CovalentlyImmobilized OHase

The distribution of the immobilized enzyme on/in the magnetic chitosancomposite particles was evaluated by labelling the OHase withfluorescein isothiocyanate (FITC) prior to the immobilization. Thefluorescent layer on the external part of the sectioned chitosancomposite particle indicated a uniform distribution of the immobilizedOHase-FITC complex on the surface of the chitosan spheres. The innerpart of the particles remained compact and did not contain immobilizedenzyme. Hence, all enzyme molecules were available to be reached by10-HSA substrate.

The invention claimed is:
 1. A recombinant oleate hydratase (OHase)immobilized on a particulate support wherein said OHase has anN-terminal His tag covalently linked to glutaraldehyde-activatedchitosan composite particles in which smaller magnetic iron oxideparticles are dispersed in a chitosan matrix.
 2. A recombinant OHaseaccording to claim 1 wherein said magnetic iron oxide particles areamino-terminated magnetic particles.